Most cancer cells display activation of phosphoinositide 3-kinase (PI3K) and the downstream enzymes AKT and TOR. Targeting the PI3K/AKT/TOR network is a promising strategy for cancer therapeutics, yet it is not clear which target profile will provide the best balance of efficacy and tolerability. Compounds that partially inhibit TOR (such as rapamycin) or compounds that inhibit both PI3K and TOR (such as PI-103) have significant limitations in efficacy and/or tolerability. A major breakthrough in this field has been the identification of novel compounds that are highly potent, selective, small molecule competitive inhibitors of TOR. Termed active-site TOR inhibitors, these compounds fully inhibit the TOR enzyme when it is present in TORC1 or TORC2, two distinct multiprotein complexes. Compared to rapamycin, an allosteric inhibitor, active-site TOR inhibitors have a broader effect on key signaling pathways and are more effective at suppressing survival of murine and human leukemia cells. Remarkably, these compounds are less immunosuppressive than rapamycin despite their greater anti-leukemic efficacy. Active-site TOR inhibitors appear equally effective in leukemia models as the less selective PI3K/TOR inhibitors, yet are considerably better tolerated in mice. Several active-site TOR inhibitors are in early stage clinical trials. The overall objective of this proposal is to refine and extend our understanding of active-site TOR inhibitors, with the ultimate goal of improving the health of cancer patients. We will focus on B cell malignancies, since active-site TOR inhibitors have dramatic effects in B cell leukemia models yet the mechanism of action remains poorly understood. The proposal has two specific aims. First, we will establish the mechanisms by which active-site TOR inhibitors trigger leukemia cell death. In this aim we will use genetic approaches to test the hypothesis that both TORC1 and TORC2 mediate survival signaling in leukemia cells. This will be accomplished using inducible Cre-mediated knockout systems and shRNA-mediated knockdown. We will then test the roles of TORC1 and TORC2 substrates in maintaining survival in leukemia cells. Second, we will define mechanisms of cellular resistance to active-site TOR inhibitors. As with any targeted molecular approach, subtypes of cancer cells display differing sensitivity to active-site TOR inhibitors. An emerging theme in drug development is the need to identify effective combinations of targeted agents. Using cell lines that do not undergo apoptosis in response to TOR inhibition, we will use candidate and global approaches to identify druggable mechanisms of resistance. We will then test combination strategies in vitro and in vivo. Identifying mechanisms of resistance and applying appropriate drug combinations will broaden the potential application of active-site TOR inhibitors.